1,3-propanediol (PDO) is a chemical building block that finds its main application in the production of polyesters. PDO can also be used as a low cost biocide and as an additive in a large number of chemical products (reviewed in (Saxena, Anand, Saran, & Isar, 2009)).
PDO can be produced by chemical synthesis using acrolein, ethylene oxide, or glycerol as starting materials. However, comparatively low product yields, harsh reaction conditions, and the production of toxic waste streams hamper cost-efficient and environmentally friendly chemical production of PDO.
PDO can also be produced by microorganisms. Natural organisms such as members of the genera Klebsiella, Citrobacter, Clostridia, and Enterobacter produce PDO during the anaerobic fermentation of glycerol where PDO synthesis serves to reoxidize excess NAD(P)H molecules produced during the conversion of glycerol into the glycolytic intermediate dihydroxyacetone phosphate. The natural biosynthesis pathway of PDO consists of a vitamin B12-dependent glycerol dehydratase which converts glycerol into 3-hydroxypropionaldehyde (3-HPA), and a 1,3-propandiol oxidoreductase which converts 3-HPA into PDO. Glycerol dehydratase and PDO oxidoreductase encoding genes are commonly grouped in an operon together with genes that encode the dehydratase reactivation factor and genes encoding enzymes for glycerol assimilation (Saxena, Anand, Saran, & Isar, 2009).
Recent approaches aim at the production of PDO from glucose by using genetically engineered microorganisms and preferentially Escherichia coli (Emptage, Haynie, Laffend, Pucci, & Whited, 2000) (Laffend, Nagarajan, & Nakamura, 1995). E. coli is not naturally capable of producing PDO. This organism was equipped with enzymes that enhance both the production of glycerol (GPD1, GPP2 of Saccharomyces cerevisiae), and the conversion of glycerol into PDO (dhaB1-3, orfZ, orfX of Klebsiella pneumoniae). It was found that its natural NADP-dependent alcohol dehydrogenase, YqhD, was capable of converting 3-HPA into PDO rendering the expression of an additional PDO oxidoreductase (e.g. dhaT) optional and even somewhat less beneficial. In addition, all genes responsible for glycerol assimilation were deleted in the production strain. The attenuation of the phosphoenolpyruvate (PEP)-dependent phosphotransferase system, and the attenuation of glyceraldehyde-3-phosphate dehydrogenase activity further increased PDO yield and productivities. This technology is currently exploited by DuPont who announced productivities of 3.5 g/Lh, final product titers of 135 g/L and carbon yields of 51% (on weight basis) in 2003 (Nakamura & Whited, 2003).
One significant drawback of this technology is the use of the vitamin B12-dependent glycerol dehydratase enzyme for PDO biosynthesis which requires supplementation of the fermentation broth with expensive vitamin B12. In addition, PDO biosynthetic pathways that employ glycerol as an intermediate depend on the utilization of fermentable sugars or glycerol as the starting material. The use of alternative carbon sources such as short and medium chain organic acids alone or in co-fermentations with sugars requires significant gluconeogenic activity therefore rendering PDO synthesis inefficient and limiting the spectrum of potential raw materials. The development of PDO-yielding pathways with entry points other than glycerol can therefore strongly contribute to increase product yield on sugars, reduce production costs by avoiding vitamin B12 dependent enzymes, and/or increase metabolic flexibility to adapt PDO production organisms to a larger panel of starting materials.
Recently, a pathway was disclosed (WO2012/004247) that describes production of PDO departing from oxaloacetate, and which proceeds through the amination of oxaloacetate to yield aspartate, the transformation of aspartate into homoserine, the deamination of homoserine to yield 2-oxo-4-hydroxybutyrate (OHB), and the conversion of OHB into PDO via 2-oxo-4-hydroxybutyratedecarboxylase and 1,3-propanediol dehydrogenase. The disclosed invention employs naturally available enzymes to build up the required reaction sequence. The theoretical PDO yield on glucose for this pathway equals the yield of PDO production from glucose via glycerol. However, since this pathway employs two transamination steps this theoretical yield will only be attained if the amino group could be entirely recycled in the transamination reactions and if NADPH-consuming de novo synthesis of glutamate would not be required. This is not very likely to occur.
The present invention represents an alternative to the existing technology by producing PDO from the organic acid malate without the need of gluconeogenic activity, without the need for metabolically costly transamination reactions, and without employing vitamin B12-dependent enzymes. In particular, the invention comprises the production of PDO from 2,4-dihydroxybutyric acid (DHB) via a non-natural synthetic pathway, and the functional expression of this pathway in a host organism to zymotically produce PDO from, for example, sugars such as glucose.